Project Summary Arch Innotek proposes to develop a sustainable, cost-effective fermentation process to produce lutein using engineered yeast. Lutein is an important antioxidant compound that can slow the progression of vision loss caused by age-related macular degeneration (AMD), cataracts, and retinitis pigmentosa. Aside from protecting eyes, dietary intake of lutein can reduce risk of skin disorders, several types of cancer including colon or breast cancer, type II diabetes and coronary heart disease. Current lutein manufacturing is mainly by extraction from plant materials (e.g., the petals of marigold flowers), which contains lutein in the ester form and is poorly absorbed by human body. Besides, this is an expensive, labor-intensive and land-demanding process and most lutein resource in US relies on imports. The high price and market demand drive companies to seek alternative sources. Based on our preliminary studies and patent applications, Arch Innotek will apply metabolon engineering and fermentation optimizations (guided by metabolic analyses) to engineer a non- model GRAS (Generally Recognized as Safe) yeast, which will lead to a safe and cost-effective production process for manufacturing high bioavailability free-lutein. Arch Innotek is the first company focused on de novo lutein production by yeast fermentation process, and our preliminary studies have successfully created producing-lutein strain. In this proposal, we are proposing innovative approaches to improve yeast productivity to achieve the break-even point (titer~1.5 g/L). We will perform metabolon engineering of lutein biosynthetic pathway to enhance fluxes towards lutein away from the competitive pathway. Moreover, 13C-based metabolic analysis will be used to characterize metabolic responses in engineered yeasts and identify hidden factors (e.g., rate limiting enzyme nodes, cofactor balances, competing pathways, and nutrient bottlenecks) for further genetic modification and fermentation optimizations. After laboratory successes, future strain engineering, scale-up and downstream purification process will be undertaken to demonstrate the technology?s value for inexpensive commercial production (Phase II). Our free-lutein will have better absorption and bioavailability when used as nutritional supplements and other health products, leading to good customer acceptance and market profitability. We will develop strategic partnerships with established companies via supply agreements, co-promotion, strategic investments, or technology licensing at certain market segments. Our product could not only have dramatic impact on lutein users, but also create numerous domestic biotechnology high paying jobs. The established biotechnology from this study may also provide broad impact/guidelines on manufacturing other carotenoid products.